Mechanism of G551D-CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Potentiation by a High Affinity ATP Analog

Bompadre, Silvia G.; Li, Min; Hwang, Tzyh Chang

doi:10.1074/jbc.m709417200

Cited by 41 publications

(48 citation statements)

References 29 publications

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“…1A shows the effect of 1 mM dATP and 1 mM 3-dATP on G551D-CFTR currents pre-activated with ATP plus PKA. Upon removal of ATP plus PKA, the current remains unchanged as reported previously (14,15). Application of 1 mM dATP enhanced G551D-CFTR currents by 7.7 Ϯ 0.6-fold (n ϭ 12), and the potentiation effect was readily reversible when dATP was removed.…”

Section: G551d-cftr Gating By 2ј-and 3ј-deoxy-atp-supporting

confidence: 53%

“…The doseresponse relationship for P-dATP (Fig. 2D) demonstrates that the apparent affinity for P-dATP is indeed similar to that of P-ATP on G551D (15). The effect of P-dATP in WT channels is shown in supplemental Fig.…”

Section: G551d-cftr Gating By 2ј-and 3ј-deoxy-atp-mentioning

confidence: 98%

“…Site-directed Mutagenesis-All mutation constructs were generated using the QuikChange XL kit (Stratagene, La Jolla, CA) as described previously (14,15). The entire CFTR cDNA was sequenced to confirm the mutation (DNA core, University of Missouri).…”

Section: Methodsmentioning

confidence: 99%

“…The mutant CFTR protein can traffic normally to the apical membrane and is normally phosphorylated by cAMP-dependent protein kinase (11)(12)(13), but it has an extremely low open probability (P o ), ϳ100 times smaller than that of WT (14). Although ATP fails to open G551D channels, the high affinity ATP analog N 6 -(2-phenylethyl)-ATP (P-ATP) can enhance the G551D-CFTR activity by ϳ7-fold mainly by increasing the open time (15). Interestingly, 2Ј-deoxy-ATP (dATP), an ATP analog with a similar affinity to ATP, has been reported to increase the activity of G551D to a similar extent as P-ATP (16).…”

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confidence: 99%

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Potentiation of Disease-associated Cystic Fibrosis Transmembrane Conductance Regulator Mutants by Hydrolyzable ATP Analogs

Miki

Zhou

et al. 2010

Journal of Biological Chemistry

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The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP-binding cassette transporter superfamily. CFTR is gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBDs), which dimerize in the presence of ATP to form two ATP-binding pockets (ABP1 and ABP2). Mutations reducing the activity of CFTR result in the genetic disease cystic fibrosis. Two of the most common mutations causing a severe phenotype are G551D and ⌬F508. Previously we found that the ATP analog N 6 -(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by ϳ7-fold. Here we show that 2-deoxy-ATP (dATP), but not 3-deoxy-ATP, increases the activity of G551D-CFTR by ϳ8-fold. We custom synthesized N 6 -(2-phenylethyl)-2-deoxy-ATP (P-dATP), an analog combining the chemical modifications in dATP and P-ATP. This new analog enhances G551D current by 36.2 ؎ 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Interestingly, P-dATP completely rectified the gating abnormality of ⌬F508-CFTR by increasing its activity by 19.5 ؎ 3.8-fold through binding to both ABPs. This result highlights the severity of the gating defect associated with ⌬F508, the most prevalent disease-associated mutation. The new analog P-dATP can be not only an invaluable tool to study CFTR gating, but it can also serve as a proof-of-principle that, by combining elements that potentiate the channel activity independently, the increase in chloride transport necessary to reach a therapeutic target is attainable. The cystic fibrosis transmembrane conductance regulator (CFTR)2 chloride channel is a major player in salt and water transport across epithelia. Like all members of the ATPbinding cassette (ABC) family, CFTR has two nucleotide-binding domains (NBDs), which contain the Walker A and B motifs and the highly conserved signature sequence. A regulatory (R) domain, unique to CFTR, needs to be phosphorylated by protein kinase A (PKA) for the channel to function. Experimental evidence suggests that the two NBDs of CFTR dimerize in a head-to-tail configuration (1), as in other ABC transporters, forming two ATP-binding pockets (ABPs) with two ATP molecules sandwiched in the interface. ABP1 is formed by the Walker A and B motifs of NBD1, and the signature sequence of NBD2 and ABP2 is formed by the Walker A and B motifs of NBD2 and the signature sequence of NBD1. Interestingly, two ABPs of CFTR assume distinct functional roles in controlling CFTR gating. Zhou et al. (2) showed that ABP2 is the site critical for channel opening by ATP, whereas the role of ABP1 is limited to help with the stabilization of the open channel conformation. Furthermore, although ABP1 seems unable to hydrolyze ATP, ATP hydrolysis at NBD2 is associat...

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Section: G551d-cftr Gating By 2ј-and 3ј-deoxy-atp-supporting

confidence: 53%

Section: G551d-cftr Gating By 2ј-and 3ј-deoxy-atp-mentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Potentiation of Disease-associated Cystic Fibrosis Transmembrane Conductance Regulator Mutants by Hydrolyzable ATP Analogs

Miki

Zhou

et al. 2010

Journal of Biological Chemistry

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“…Many compounds ( Amaral and Kunzelmann, 2007 ) and ATP analogues ( Cai et al, 2006 ;Bompadre et al, 2007 ) have been found to potentiate the activity of G551D-CFTR channels, but none of them could increase the activity of G551D-CFTR to WT levels. The binding site for only a very limited number of these CFTR " potentiatiors " has been identifi ed ( Bompadre et al, 2008 ). Interestingly, the 20-fold increase of the G551D-CFTR current by Cd 2+ is by far the most effective potentiation demonstrated for this disease-associated mutant.…”

Section: Pathophysiological Implicationsmentioning

confidence: 99%

Mutations at the Signature Sequence of CFTR Create a Cd2+-gated Chloride Channel

Wang

Bompadre

et al. 2008

The Journal of General Physiology

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The canonical sequence LSGGQ, also known as the signature sequence, defi nes the adenosine triphosphate (ATP)-binding cassette transporter superfamily. Crystallographic studies reveal that the signature sequence, together with the Walker A and Walker B motifs, forms the ATP-binding pocket upon dimerization of the two nucleotide-binding domains (NBDs) in a head-to-tail confi guration. The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G551D) in cystic fi brosis transmembrane conductance regulator (CFTR) results in a severe phenotype of cystic fi brosis. We previously showed that the G551D mutation completely eliminates ATP-dependent gating of the CFTR chloride channel. Here, we report that micromolar [Cd 2+ ] can dramatically increase the activity of G551D-CFTR in the absence of ATP. This effect of Cd 2+ is not seen in wild-type channels or in G551A. Pretreatment of G551D-CFTR with the cysteine modifi cation reagent 2-aminoethyl methane thiosulfonate hydrobromide protects the channel from Cd 2+ activation, suggesting an involvement of endogenous cysteine residue(s) in mediating this effect of Cd 2+ . The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR ' s NBD1, show robust response to Cd 2+ . On the other hand, negligible effects of Cd 2+ were seen with T547C, Q552C, and R553C, indicating that a specifi c region of the signature sequence is involved in transmitting the signal of Cd 2+ binding to the gate. Collectively, these results suggest that the effect of Cd 2+ is mediated by a metal bridge formation between yet to be identifi ed cysteine residue(s) and the engineered aspartate or cysteine in the signature sequence. We propose that the signature sequence serves as a switch that transduces the signal of ligand binding to the channel gate.

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G551D‐CFTR needs more bound actin than wild‐type CFTR to maintain its presence in plasma membranes

Trouvé

Kerbiriou

Teng

et al. 2015

Cell Biology International

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Cystic Fibrosis is due to mutations in the CFTR gene. The missense mutation G551D (approx. 5% of cases) encodes a CFTR chloride channel with normal cell surface expression but with an altered chloride channel activity, leading to a severe phenotype. Our aim was to identify specific interacting proteins of G551D-CFTR which could explain the channel defect. Wild-type CFTR (Wt-CFTR) was co-immunoprecipitated from stably transfected HeLa cells and resolved by 2D gel electrophoresis. Among the detected spots, one was expressed at a high level. Mass Spectrometry revealed that it corresponded to actin which is known to be involved in the CFTR's channel function. To assess whether actin could be involved in the altered G551D-CFTR function, its basal expression was studied. Because actin expression was the same in wt- and in G551D-CFTR expressing cells, its interaction with both wt- and G551D-CFTR was studied by co-immunoprecipitation, and we found that a higher amount of actin was bound onto G551D-CFTR than onto Wt-CFTR. The role of actin upon wt- and G551D-CFTR function was further studied by patch-clamp experiments after cytochalasin D treatment of the cells. We found a decrease of the very weak currents in G551D-CFTR expressing cells. Because a higher amount of actin is bound onto G551D-CFTR than onto Wt-CFTR, it is likely to be not involved in the mutated CFTR's defect. Nevertheless, because actin is necessary to maintain the very weak global currents observed in G551D-CFTR expressing HeLa cells, we conclude that more actin is necessary to maintain G551D-CFTR in the plasma membrane than for Wt-CFTR.

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Mechanism of G551D-CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Potentiation by a High Affinity ATP Analog

Cited by 41 publications

References 29 publications

Potentiation of Disease-associated Cystic Fibrosis Transmembrane Conductance Regulator Mutants by Hydrolyzable ATP Analogs

Potentiation of Disease-associated Cystic Fibrosis Transmembrane Conductance Regulator Mutants by Hydrolyzable ATP Analogs

Mutations at the Signature Sequence of CFTR Create a Cd2+-gated Chloride Channel

G551D‐CFTR needs more bound actin than wild‐type CFTR to maintain its presence in plasma membranes

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